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Updated: Dec 27, 2025

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HAMA: a multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes
Aleksa Stanišić1, Annika Hüsken1, Hajo Kries1
1Independent Junior Research Group Biosynthetic Design of Natural Products , Leibniz Institute for Natural Product Research and Infection Biology e.V. , Hans Knöll Institute (HKI Jena) , Beutenbergstr. 11a , 07745 Jena , Germany .
Abstract:
Adenylation enzymes selecting substrates for ribosomal and nonribosomal protein and peptide biosynthesis have been popular targets of enzyme engineering. Previous standard assays for adenylation specificity have been cumbersome and failed to reflect the competition conditions inside a cell because they measure substrates one at a time. We have developed an adenylation assay based on hydroxamate quenching and LC-MS/MS detection of hydroxamate products testing dozens of competing amino acid substrates in parallel. Streamlined specificity profiling of adenylation enzymes will facilitate engineering and directed evolution of ribosomal and nonribosomal peptide synthesis.
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